Anti-porcine reproductive and respiratory syndrome virus antibody or antigen binding fragment thereof and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-porcine reproductive and respiratory syndrome virus antibody or an antigen binding fragment thereof and application thereof. The antibody or the antigen binding fragment thereof can specifically recognize and bind the porcine reproductive and respiratory syndrome virus or the GP4 protein thereof, and has good affinity with the porcine reproductive and respiratory syndrome virus or the GP4 protein thereof; the compound can be used for preparing products for diagnosing, preventing and / or treating porcine reproductive and respiratory syndrome virus infection or diseases caused by porcine reproductive and respiratory syndrome virus infection, detecting existence or level of porcine reproductive and respiratory syndrome virus or GP4 protein thereof in a sample or screening drugs for preventing and / or treating porcine reproductive and respiratory syndrome virus infection or diseases caused by porcine reproductive and respiratory syndrome virus infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to antibodies against porcine reproductive and respiratory syndrome virus or their antigen-binding fragments and their applications. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). The main symptoms are respiratory illness and reproductive disorders in pregnant sows, such as abortion and stillbirth. Because the surface symptom is a bluish-purple discoloration of the skin around the ears, it is also known as "blue ear disease." PRRS is a relatively new and widespread swine disease globally, and it is a causative factor leading to reproductive and respiratory disorders in pig herds. First reported in Europe and the United States in the 1990s, it has since had a serious impact on the global pig industry.

[0003] PRRSV belongs to the order Taurivirales, family Arteriviridae, and genus Arterivir. PRRSV is an enveloped, single-stranded, positive-sense RNA virus with a genome length of approximately 15 kb. It consists of an untranslated region (UTR) and multiple open reading frames (ORFs). There is some overlap between different ORFs of PRRSV, which is a characteristic of viruses in the Arteriviridae family. PRRSV has two genotypes: the European type (type I) and the American type (type II).

[0004] Pigs are susceptible to PRRSV, and carrier and infected pigs can be sources of infection for the disease, which can occur year-round. Therefore, it is necessary to develop antibodies against PRRSV for the detection and / or prevention of porcine reproductive and respiratory syndrome virus (PRRSV) or the diseases it causes. Summary of the Invention

[0005] The first aspect of the present invention aims to provide an antibody against porcine reproductive and respiratory syndrome virus or an antigen-binding fragment thereof.

[0006] A second aspect of the present invention is to provide a chimeric antigen receptor.

[0007] A third aspect of the present invention aims to provide a multispecific antibody or an antigen-binding fragment thereof.

[0008] The fourth aspect of this invention aims to provide biomaterials.

[0009] The fifth aspect of this invention aims to provide a method for preparing an antibody or antigen-binding fragment thereof according to the first aspect of this invention, a chimeric antigen receptor according to the second aspect, or a multispecific antibody or antigen-binding fragment thereof according to the third aspect.

[0010] The sixth aspect of this invention aims to provide a coupling.

[0011] A seventh aspect of the present invention is to provide a pharmaceutical composition.

[0012] An eighth aspect of the present invention aims to provide a diagnostic or therapeutic reagent kit.

[0013] The object of the ninth aspect of the present invention is to provide the use of the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the chimeric antigen receptor of the second aspect, the multispecific antibody or antigen-binding fragment thereof of the third aspect, the biomaterial of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

[0014] The object of the tenth aspect of this invention is to provide a method for preventing and / or treating porcine reproductive and respiratory syndrome virus infection or diseases caused thereby.

[0015] The object of the eleventh aspect of this invention is to provide a method.

[0016] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides an antibody against porcine reproductive and respiratory syndrome virus (PRRSV) or an antigen-binding fragment thereof, said antibody against PRSV or an antigen-binding fragment thereof comprising: a1) HCDR1, HCDR2 and HCDR3 included in the heavy chain variable region (VH) of the amino acid sequence shown in SEQ ID NO: 9; and / or LCDR1, LCDR2 and LCDR3 included in the light chain variable region (VL) of the amino acid sequence shown in SEQ ID NO: 10. a2) having one or more amino acid substitutions, deletions, or additions to HCDR1, HCDR2, and HCDR3 compared to those shown in a1); and / or having one or more amino acid substitutions, deletions, or additions to LCDR1, LCDR2, and LCDR3 compared to those shown in a1).

[0017] In some implementations, the CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

[0018] In some embodiments, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment comprises: b1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 13, HCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 15; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 16, LCDR2 having the amino acid sequence AAT, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 17; b2) VH comprising the following three CDRs: having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 shown in b1); and / or VL comprising the following three CDRs: having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 shown in b1); The CDR is defined according to the IMGT numbering system.

[0019] In some embodiments, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment comprises: c1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 18, HCDR2 having the amino acid sequence shown in SEQ ID NO: 19, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 20; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 17; or c2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 shown in c1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 shown in c1); The CDR is defined according to the Kabat numbering system.

[0020] In some embodiments, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment comprises: d1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 23, HCDR2 having the amino acid sequence shown in SEQ ID NO: 24, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 20; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 17; or d2) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 shown in d1); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 shown in d1); The CDR is defined according to the Chothia numbering system.

[0021] In some embodiments, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment comprises: e1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 25, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 28, LCDR2 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 30; or e2) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 shown in e1); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 shown in e1); The CDR is defined according to the Contact numbering system.

[0022] Those skilled in the art should understand that the above-mentioned amino acid substitutions are conservative substitutions.

[0023] In some embodiments, the heavy chain variable region of the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment further includes the framework region of the heavy chain variable region.

[0024] In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region of immunoglobulins derived from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese, or a mutant thereof; further, it includes the framework region of the heavy chain variable region of mouse immunoglobulins, or a mutant thereof.

[0025] In some embodiments, the light chain variable region of the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment further includes the framework region of the light chain variable region.

[0026] In some embodiments, the framework region of the light chain variable region includes the framework region of the light chain variable region of immunoglobulins derived from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese, or a mutant thereof; further, it includes the framework region of the light chain variable region of mouse immunoglobulins, or a mutant thereof.

[0027] In some embodiments, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment comprises: The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0028] In some embodiments, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0029] In some embodiments, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

[0030] In some embodiments, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

[0031] In some embodiments, the heavy chain constant region includes a heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2 (e.g., IgG2a, IgG2b), IgG3, IgG4, or IgM immunoglobulin.

[0032] In some embodiments, the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0033] In some embodiments, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins.

[0034] In some embodiments, the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0035] In some embodiments, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0036] In some embodiments, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment may include, but is not limited to, monoclonal antibodies, nanobodies, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, single-chain Fv (scFv), dsFv, or Fd fragments.

[0037] In some embodiments, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment specifically binds to the porcine reproductive and respiratory syndrome virus GP4 protein.

[0038] In some embodiments, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment specifically binds to the porcine reproductive and respiratory syndrome virus GP234 complex.

[0039] In some embodiments, the GP234 complex includes GP2 protein, GP3 protein, and GP4 protein.

[0040] A second aspect of the invention provides a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof from the first aspect of the invention.

[0041] A third aspect of the invention provides a multispecific antibody or an antigen-binding fragment thereof comprising two or more (e.g., three or four) antigen-binding domains, wherein one antigen-binding domain comprises the antibody or an antigen-binding fragment thereof of the first aspect of the invention.

[0042] A fourth aspect of the invention provides a biological material comprising any one of n1)-n9): n1) A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the first aspect of the present invention, a chimeric antigen receptor of the second aspect, or a multispecific antibody or antigen-binding fragment thereof of the third aspect; n2) An expression cassette containing the nucleic acid molecule described in n1); n3) A carrier containing the nucleic acid molecule described in n1); n4) A carrier containing the expression box described in n2); n5) A cell containing the nucleic acid molecules described in n1); n6) Cells containing the expression cassette described in n2); n7) Cells containing the carrier described in n3); n8) Cells containing the carrier described in n4); n9) Cells comprising an antibody or antigen-binding fragment thereof of the first aspect of the present invention, a chimeric antigen receptor of the second aspect, or a multispecific antibody or antigen-binding fragment thereof of the third aspect; None of the cells described in n5)-n9) contain reproductive material.

[0043] Those skilled in the art will understand that nucleotides in nucleic acid molecules can be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0044] In some embodiments, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the present invention and a nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the present invention.

[0045] In some embodiments, the nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment of the first aspect of the invention comprises: SEQ ID NO: 11, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0046] In some embodiments, the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment of the first aspect of the invention comprises: SEQ ID NO: 12, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0047] In some embodiments, any one of the vectors n3)-n4) is an expression vector. In some embodiments, the expression vector includes a eukaryotic expression vector and / or a prokaryotic expression vector. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0048] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0049] In some embodiments, any of the cells (n5)-n9) can be host cells conventionally used in the art, as long as the expression vector can stably express the carried nucleic acid molecule as the antibody or its antigen-binding fragment, chimeric antigen receptor, or multispecific antibody or its antigen-binding fragment of the present invention. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0050] In some embodiments, any of the cells (n5)-n9) can be immune cells. In some embodiments, the immune cells may include, but are not limited to, T cells, NK cells, DC cells, and macrophages. In these embodiments, the immune cells may express the chimeric antigen receptor (i.e., modified immune cells) described above in this invention.

[0051] A fifth aspect of the present invention provides a method for preparing an antibody or antigen-binding fragment thereof from the first aspect of the present invention, a chimeric antigen receptor from the second aspect of the present invention, or a multispecific antibody or antigen-binding fragment thereof from the third aspect of the present invention, obtained by culturing cells from the fourth aspect of the present invention.

[0052] A sixth aspect of the invention provides a conjugate comprising an antibody or antigen-binding fragment thereof from the first aspect of the invention or a multispecific antibody or antigen-binding fragment thereof from the third aspect of the invention; and a conjugation portion.

[0053] In some implementations, the coupling portion may include, but is not limited to, a detectable marker or a therapeutic agent.

[0054] In some embodiments, the detectable marker can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, chemical, etc. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention using linkers of different lengths to reduce potential steric hindrance.

[0055] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, biotin, etc.

[0056] In some embodiments, the therapeutic agent may include, for example, but not limited to, drugs for the prevention and / or treatment of porcine reproductive and respiratory syndrome virus infection or diseases caused by it.

[0057] In some embodiments, the coupling portion is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl groups.

[0058] A seventh aspect of the invention provides a pharmaceutical composition comprising: an antibody or antigen-binding fragment thereof of the first aspect of the invention, a chimeric antigen receptor of the second aspect, a multispecific antibody or antigen-binding fragment thereof of the third aspect, a biological material of the fourth aspect, or a conjugate of the sixth aspect; and a pharmaceutically acceptable carrier.

[0059] In some embodiments, the pharmaceutical composition may also include additional pharmaceutically active agents.

[0060] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, such as a drug capable of preventing and / or treating porcine reproductive and respiratory syndrome virus infection or diseases caused by it.

[0061] In some embodiments, the antibody or its antigen-binding fragment, chimeric antigen receptor, multispecific antibody or its antigen-binding fragment, biomaterial or conjugate, and the additional pharmaceutically active agent are provided as separate components or as mixed components.

[0062] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[0063] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, etc.

[0064] An eighth aspect of the present invention provides a diagnostic or therapeutic kit comprising: an antibody or antigen-binding fragment thereof of the first aspect of the present invention, a chimeric antigen receptor of the second aspect, a multispecific antibody or antigen-binding fragment thereof of the third aspect, a biomaterial of the fourth aspect, a conjugate of the sixth aspect, or a pharmaceutical composition of the seventh aspect.

[0065] In some embodiments, the kit may also include instructions and / or a drug delivery device.

[0066] In some embodiments, the kit can be used to diagnose porcine reproductive and respiratory syndrome virus (PRRSV) infection or diseases caused by it, detect the presence or level of PRRSV or its GP4 protein in a sample, or screen for drugs used to prevent and / or treat PRRSV infection or diseases caused by it.

[0067] In some embodiments, the kit can be used to prevent and / or treat porcine reproductive and respiratory syndrome virus infection or diseases caused by it.

[0068] A ninth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the second aspect, the multispecific antibody or antigen-binding fragment thereof of the third aspect, the biomaterial of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect in any one of d1)-d6): d1) Prepare products for the diagnosis of porcine reproductive and respiratory syndrome virus infection or diseases caused by it; d2) Prepare products for the prevention and / or treatment of porcine reproductive and respiratory syndrome virus infection or diseases caused by it; d3) Prepare products for detecting the presence or level of porcine reproductive and respiratory syndrome virus or its GP4 protein in samples; d4) Detect the presence or level of porcine reproductive and respiratory syndrome virus or its GP4 protein; d5) Prepare products for drug screening, wherein the drugs are used to prevent and / or treat porcine reproductive and respiratory syndrome virus infection or diseases caused by it; d6) Drug screening, wherein the drug is used to prevent and / or treat porcine reproductive and respiratory syndrome virus infection or diseases caused thereby.

[0069] In some implementations, the applications described in c4) and c6) do not involve the diagnosis or treatment of diseases.

[0070] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excretions of the subject.

[0071] In some embodiments, the body fluid includes at least one of blood and lymph.

[0072] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0073] In some embodiments, the excrement includes at least one of urine, feces, and tears.

[0074] In some implementations, the test subject includes mammals such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0075] In some implementations, the test subject includes pigs.

[0076] In a tenth aspect of the present invention, a method for preventing and / or treating porcine reproductive and respiratory syndrome virus infection or diseases caused therefrom is provided, the method comprising administering to a subject in need an effective amount of an antibody or antigen-binding fragment thereof of the first aspect of the present invention, a chimeric antigen receptor of the second aspect, a multispecific antibody or antigen-binding fragment thereof of the third aspect, a biomaterial of the fourth aspect, a conjugate of the sixth aspect, or a pharmaceutical composition of the seventh aspect.

[0077] The object of the eleventh aspect of the present invention is to provide a method comprising, under conditions that allow the antibody or antigen-binding fragment of the first aspect of the present invention or the multispecific antibody or antigen-binding fragment of the third aspect of the present invention to form a complex with the porcine reproductive and respiratory syndrome virus GP4 protein, contacting a sample with the antibody or antigen-binding fragment of the first aspect of the present invention or the multispecific antibody or antigen-binding fragment of the third aspect of the present invention, and detecting the formation of the complex; The method is used for any one of f1)-f3): f1) Diagnose porcine reproductive and respiratory syndrome virus infection or the disease it causes; f2) Detect the presence or level of porcine reproductive and respiratory syndrome virus or its GP4 protein in the sample; f3) Screening for drugs for the prevention and / or treatment of porcine reproductive and respiratory syndrome virus infection or diseases caused by it.

[0078] In some embodiments, the sample is the sample of the ninth aspect of the present invention.

[0079] In this invention, the porcine reproductive and respiratory syndrome virus includes the NADC30 strain.

[0080] In this invention, the amino acid sequence of the GP2 protein includes: SEQ ID NO:1, or an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0081] In this invention, the amino acid sequence of the GP3 protein includes: SEQ ID NO:3, or an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0082] In this invention, the amino acid sequence of the GP4 protein includes: amino acids 18-178 of SEQ ID NO:4 and SEQ ID NO:33, amino acids 1-178 of SEQ ID NO:33, or amino acid sequences having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with them.

[0083] In this invention, the diseases caused by the porcine reproductive and respiratory syndrome virus infection include porcine reproductive and respiratory syndrome.

[0084] The beneficial effects of this invention are: This invention provides an antibody against porcine reproductive and respiratory syndrome virus (PRRSV) or its antigen-binding fragment, which can specifically recognize and bind to PRRSV or its GP4 protein and has good affinity for it; it can be used to prepare products for the diagnosis, prevention and / or treatment of PRRSV infection or diseases caused by PRRSV, detection of the presence or level of PRRSV or its GP4 protein in samples, or screening for drugs to prevent and / or treat PRRSV infection or diseases caused by PRRSV. Attached Figure Description

[0085] Figure 1 The results of GP4-6 antibody subtype assay are shown.

[0086] Figure 2 The results of SDS-PAGE electrophoresis identification of the genetically engineered antibody (GP4-6 antibody) are shown.

[0087] Figure 3 The results of flow cytometry validation of the genetically engineered antibody (GP4-6 antibody) are shown.

[0088] Figure 4 The results of affinity assays for the genetically engineered antibody (GP4-6 antibody) and the GP234 complex are shown.

[0089] Figure 5The binding results of the genetically engineered antibody (GP4-6 antibody) to the GP234 complex are shown. Detailed Implementation

[0090] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0091] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0092] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0093] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0094] As used in this article, "antibody" refers to a globulin produced by plasma cells, which are formed from the proliferation and differentiation of B lymphocytes in response to antigen stimulation. Antibodies specifically bind to the corresponding antigens and mediate immune effects. They are mainly found in serum and body fluids and are important immune molecules mediating humoral immunity. Antibodies can encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific, trispecific, or tetraspecific antibodies), single-chain molecules, and antigen-binding fragments. The chemical basis of antibodies is immunoglobulin (Ig).

[0095] As used herein, the term "monoclonal antibody" refers to antibodies derived from a substantially homogeneous group of antibodies, meaning that, apart from possible trace amounts of variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, typically present in small quantities), the individual antibodies within the group are identical and / or bind to the same epitopes. Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0096] As used herein, the term "multispecific antibody" is used in its broadest sense to encompass antibodies exhibiting multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies, and trispecific antibodies, antibody fragments covalently or non-covalently linked, etc.

[0097] The terms “full-length antibody” and “intact antibody” as used herein are used interchangeably to refer to antibodies that are structurally similar to natural antibodies. “Natural antibody” refers to a naturally occurring immunoglobulin molecule. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions, CH). From the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain constant region). The heavy chain of an antibody can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and can be further subdivided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody, based on the amino acid sequence of its constant domain, can be one of two types: the κ (kappa) light chain and the λ (lambda) light chain.

[0098] Within the light and heavy chains, variable and constant regions are linked by a "J" region containing approximately 12 or more amino acid residues, and the heavy chain also contains a "D" region containing approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0099] The term "Fd fragment" as used herein refers to an antibody fragment consisting of VH and CH1 domains. The term "dAb fragment" as used herein refers to an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 (1989)). The term "Fab fragment" as used herein refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains. The term "F(ab')2 fragment" as used herein refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region. The term "Fab' fragment" as used herein refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (composed of VH and CH1 domains). The term "Fab'-SH" as used herein refers to a Fab fragment containing free thiol groups.

[0100] As used in this article, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0101] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0102] As used herein, the term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain involved in the binding of the antigen-binding molecule to the antigen. The variable regions (VH and VL) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR1-4) and three hypervariable regions (HVR1-3), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL domain is sufficient to confer antigen-binding specificity. The three HVRs within the VH and VL together constitute the antigen-binding site of Ig, which can bind complementary to the corresponding antigenic epitope; therefore, the HVRs are also called complementarity-determining regions (CDRs), denoted as CDR1, CDR2, and CDR3, respectively. The VH or VL chain of the antibody may further contain all or part of the constant regions of the heavy or light chain.

[0103] The CDR of the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment of the present invention is preferably determined by the IMGT, Kabat, Contact, Chothia, or AbM numbering system.

[0104] As used in this article, the term "variable" refers to the fact that certain segments of the variable region are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable region, but is concentrated in three segments called hypervariable regions (HVRs) within the variable regions of the light and heavy chains. The relatively highly conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are tightly held together by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site. Constant regions do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0105] Antibody "classes" refer to the types of constant structural domains or constant regions possessed by the antibody's heavy chain. Based on differences in heavy chain structure and antigenicity, they can be classified into five classes: μ chain, γ chain, α chain, δ chain, and ε chain. Immunoglobulins composed of different heavy and light chains are respectively called IgA, IgD, IgE, IgG, and IgM. Even within the same class of Ig, the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain differ, thus further subdividing the same class of Ig into different subclasses. For example, human IgG can be divided into IgG1–IgG4; IgA can be divided into IgA1 and IgA2. Based on differences in light chain structure and antigenicity, immunoglobulin (Ig) light chains are divided into κ (kappa) chains and λ (lambda) chains, thus classifying Ig into two types: κ type and λ type.

[0106] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0107] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0108] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0109] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0110] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0111] Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0112] Example 1. Construction of eukaryotic recombinant plasmids 1) Eukaryotic recombinant plasmid expressing GP2: The gene encoding the truncated GP2 variant of NADC30 strain (MH500776) (named GP2-209, with a secretory LYSP signal peptide (MEFGLSWVFLVALFRGVQC, SEQ ID NO:2) added to the N-terminus and a His tag added to the C-terminus) was inserted into the pCMV plasmid (brand: Youbao Biotechnology; name: pCMV-HA-C vector; catalog number: VT1065; the same below) containing the Sal gene. Between I and EcoR I, a eukaryotic recombinant plasmid expressing GP2 was obtained.

[0113] 2) Eukaryotic recombinant plasmid expressing GP3: The gene encoding the GP3 mutant of the NADC30 strain (named: GP3-M3; a Flag tag was added to the N-terminus of the GP3 mutant of the NADC30 strain (APSANSTYCFWFPLVRGNFSFELMVNYTVCPPCLTRQAAAQILEPGGSLWCRIGHDRCEESDHDELEFAVPPGLSVEGRLTSVYAWLAFLSFSYTAQFHPEIFGIGNVSEVYVDIQNQLICAVHDGLNATLPSHDNITAVYQSYYQHQVDGGNWFHLEAARPAFSSALVANVSAFLRRSPVSRVSVRVFQTLRPTPPAQQVPLSSKTSAVLGIATRPLRRFAKSLNVARR, SEQ ID NO:3), and a secretory LYSP signal peptide was added to the N-terminus of the Flag tag) was inserted between Sal I and EcoR I of the pCMV plasmid to obtain the eukaryotic recombinant plasmid expressing GP3.

[0114] 3) Eukaryotic recombinant plasmid expressing GP4: The gene encoding GP4 of the NADC30 strain (with a Strep tag added to the N-terminus of GP4 (SQAFACKPCFSSSLSDIKTNTTSAASSVVLQNIGCLRHSNPSPAAFRKIPQCRAAIGTPVYITVTANVTDENYLHSSDLLMLSSCLFYASEMSEKGFKVIFGNVSGIVAVCVNFTSYVQHVREFTQRSLVVDHV, SEQ ID NO:4) of the NADC30 strain, and a secretory LYSP signal peptide added to the N-terminus of the Strep tag) was inserted between Sal I and EcoR I of the pCMV plasmid to obtain the eukaryotic recombinant plasmid expressing GP4.

[0115] 2. Large-scale preparation of eukaryotic recombinant plasmids Take 1 μg of the eukaryotic recombinant plasmids expressing GP2 / GP3 / GP4 obtained in step 1 and mix them with 100 μL of Top10 competent cells (purchased from Qingke Biotechnology). Incubate on ice for 15 minutes. After heat shock at 42℃ for 90 seconds, incubate on ice for 5 minutes, add antibiotic-free liquid LB medium, and incubate at 37℃ and 220 rpm for 40 minutes. After incubation, centrifuge at 2000×g for 5 minutes, discard most of the supernatant, resuspend the competent cells in the remaining medium, and spread evenly on solid LB (Amp) medium. + Incubate in a petri dish at 37°C for 12–16 hours. After incubation, select a single, well-growing colony from the petri dish for further culture and extract plasmids according to the instructions of the Tiangen plasmid extraction kit.

[0116] 3. Expression and purification of the immunogen (GP234 complex) Take the eukaryotic recombinant plasmid (which contains: 1 mg of the eukaryotic recombinant plasmid expressing GP2 obtained in step 2, 1 mg of the eukaryotic recombinant plasmid expressing GP3 obtained in step 2, and 1 mg of the eukaryotic recombinant plasmid expressing GP4 obtained in step 2) and mix it with the transfection reagent PEI (purchased from Polyscience). After mixing, let it stand at room temperature for 5-10 minutes. Take a density of 3×10 61 L of 293F cells / mL was added dropwise to the prepared plasmid / PEI mixture while shaking, and cultured for 4 days at 130 rpm in a 37°C incubator with 5% CO2. After 4 days, the cells were harvested, and the culture was centrifuged to collect the supernatant. Cell debris was removed by filtration through a 0.45 μM filter. The supernatant was concentrated by ultrafiltration (10 kD) using a membrane encapsulation method, and then diluted with 1×PBS buffer for later use. The Ni column was equilibrated with 1×PBS buffer, and the supernatant was loaded onto the Ni column. 5-10 column volumes of the column were loaded with washing buffer (1×PBS buffer, 20 mM imidazole). After baseline equilibration, the target protein was eluted with elution buffer (1×PBS buffer, 500 mM imidazole). The eluted protein solution was filtered through a 0.22 μM filter for sterilization to obtain the GP234 complex. Protein concentration was determined using NanoDrop.

[0117] 4. Mouse Immunization Prepare 6-8 week old BALB / c mice. On days 1 and 30 of the experiment, immunize the mice with the GP234 complex purified in step 3 (intraperitoneal injection, each immunization dose is 20 µg based on the GP234 complex). For the first immunization, Freund's complete adjuvant was used as the adjuvant and CpG as the enhancer (240 µg of GP234 complex was dissolved in 600 µL PBS buffer to obtain the GP234 complex solution; CpG 4 OD was dissolved in 600 µL PBS buffer to obtain the CpG solution; the GP234 complex solution, CpG solution and 1200 µL Freund's complete adjuvant were mixed to obtain a 2.4 mL emulsion, which can be used to immunize 12 mice). For the second immunization, Freund's incomplete adjuvant was used as the adjuvant and CpG as the enhancer (240 µg of GP234 complex was dissolved in 600 µL PBS buffer to obtain the GP234 complex solution; CpG 4 OD was dissolved in 600 µL PBS). The buffer was used to obtain the CpG solution; the GP234 complex solution, CpG solution and 1200 µL Freund's incomplete adjuvant were mixed and the resulting 2.4 mL system was emulsified (which can be used to immunize 12 mice). Blood was collected from the orbital rim two weeks after the second immunization to obtain the corresponding immune serum.

[0118] 5. Antibody screening Feeder cells were prepared (the peritoneal cavity of unimmunized wild-type mice was rinsed with 0.34 M sucrose solution, cells were collected and resuspended in HAT medium, and seeded into 96-well plates, 100 μL / well) and spleen cells were prepared (the spleens of mice obtained in step 4 were ground in 1640 medium containing DNase, filtered through a 70 μm filter, treated with ACK erythrocyte lysis buffer for 2 minutes, and then resuspended). Spleen cells were mixed with Sp2 / 0 mouse myeloma cells in logarithmic growth phase at a ratio of 1:1-1:3, centrifuged at 1500 rpm for 10 minutes, the supernatant was discarded, and 1 mL of preheated 50% PEG solution was added dropwise in a 37°C water bath, followed by 40 mL of preheated 1640 medium. After centrifugation again, the supernatant was discarded, the cells were resuspended in HAT medium and the volume was increased to approximately 90 mL. The medium was then added dropwise to 96-well plates pre-coated with feeder cells (2 drops per well). Four days after fusion, half of the medium (HAT medium) was replaced. After 7-10 days, when the hybridoma cell clusters grew to a certain size, the culture supernatant was aspirated as the test antibody. The titer was tested using serum from immunized mice as a positive control and serum from unimmunized mice as a negative control to screen for positive wells.

[0119] Subcloning was performed on positive wells using a limiting dilution method: For the first subcloning, HT medium was used. After counting, 100-150 cells were seeded into 9.5 mL of medium, mixed, and added to a plate containing feeder cells. After approximately 5 days, cell counts were observed. Once the cell clusters had grown, positive clones were screened again. Subcloning was then repeated 2-3 times using 1640 complete medium until a stable antibody-secreting monoclonal hybridoma cell line was obtained. Finally, the positive cells were expanded (in 24-well plates and T25 flasks sequentially until 80% confluence), centrifuged, resuspended in 3 mL of cell cryopreservation buffer, and stored at -80°C for 24 hours after programmed cooling. Then, they were transferred to liquid nitrogen for long-term storage, thus obtaining hybridoma cells secreting the target monoclonal antibody (hybridoma cells secreting antibody GP4-6).

[0120] 6. Antibody subtype determination The test antibody (GP4-6, secreted from hybridoma cells obtained in step 5) and the positive control antibody (Mouse anti Strep II tag mAb (brand: Abclonal; catalog number: AE066)) were diluted to 5 μg / mL using 1×PBS and added to an ELISA plate for overnight coating at 4°C. A negative control (BSA protein diluted in 1×PBS, 5 μg / mL) was also included. After coating, the plate was washed three times with PBS the following day to ensure the removal of unbound antibodies. Blocking buffer was added, and the plate was blocked at room temperature for 1 hour to prevent non-specific binding. After blocking, biotin-labeled IgG1, IgG2a, IgG2b, and IgG3 antibodies were added at a concentration of 10 μg / mL, and the plate was incubated at room temperature for 2 hours. After incubation, the plate was washed five times with PBST to remove unbound labeled antibodies. HRP-labeled streptavidin was then added, and the plate was incubated at room temperature for 1 hour. Finally, TMB chromogenic substrate was added, and the reaction was allowed to proceed until sufficient color development was achieved. The OD450 was then measured using a microplate reader to determine the antibody subtype. The results are as follows Figure 1 As shown: This antibody (GP4-6) is the IgG2a subtype.

[0121] 7. Antibody gene extraction Based on the subtype identification results, appropriate light chain primers (VL-F, and Igκ-R / Igλ-R) and heavy chain primers (VH-F, and IgA-R / IgE-R / IgM-R / IgG1-R / IgG2A-R / IgG2B-R / IgG3-R) were selected for PCR amplification. The amplified fragments were separated by 1.5% agarose gel electrophoresis, and the light and heavy chain amplification products were recovered separately. DNA was recovered using an agarose gel DNA purification and recovery kit, and the purified products were sent to the company for sequencing. The downstream primers of the PCR amplification system were used for sequencing. The heavy chain primers are as follows: VH-F: SARGTNMAGCTGSAGSAGTC (SEQ ID NO: 5), IgG2A-R: CTTGACCAGGCATCCTAGAGTCA (SEQ ID NO: 6), IgA-R: GATGGTGGGATTTCTCGCAGACTC (SEQ ID NO: 37), IgE-R: TAAGGGGTAGAGCTGAGGGTTCCTG (SEQ ID NO: 6) NO:38), IgM-R: GACATTTGGGAAGGACTGACTCTC (SEQ ID NO:39), IgG1-R: ATAGACAGATGGGGGTGTCGTTTTGGC (SEQ ID NO:40), IgG2B-R: AGGGGCCAGTGGATAGACTGATGG (SEQ ID NO:41), IgG3-R: AGGGACCAAGGGATAGACAGATGG (SEQ ID NO:41) NO:42); the light chain primers are as follows: VL-F: GAYATTGTGMTSACMCARWCTMCA (SEQ ID NO: 7), Igκ-R: GGATACAGTTGGTGCAGCATC (SEQ ID NO: 8), Igλ-R: GACATTCAGCTGACCCAGTCTCCA (SEQ ID NO: 43).

[0122] Next, the sequencing results were compared with the mouse genome in the NCBI database to determine the signal peptide sequences of the light and heavy chains. Based on this result, new primers were designed to amplify the entire CDS, and the DNA was purified again using an agarose gel DNA purification and recovery kit. The recovered product was sent to the company for sequencing, and sequencing was performed using the upstream and downstream primers of the PCR amplification system to finally determine the full-length sequences of the variable regions of the light and heavy chains. The amino acid / nucleotide sequences of the variable region and CDR of the antibody (GP4-6) are shown in Table 1.

[0123] Table 1. Amino acid / nucleotide sequence of the variable region and CDR of antibody (GP4-6)

[0124] 8. Expression and purification of genetically engineered antibodies

[0125] Antibody concentration was performed using a 30 kDa concentration column. Depending on the concentration effect, the supernatant was removed and the medium was repeatedly changed. Purity and concentration were determined using SDS-PAGE. After adding protein loading, the sample was boiled and electrophoresed in a gel at a rate of 5 μg. After staining, the sample was destained and photographed to assess antibody purity and concentration. Results are as follows: Figure 2 As shown, the sizes of the heavy and light chains of the antibody are consistent with the expected results, verifying the successful expression and purification of the antibody.

[0126] 9. Streaming Cytometry Verification Construct the GP2-Flag, GP3-Flag, and GP4-Flag plasmids, as follows: respectively add GP2-Flag (MKWGLCRAFLTKSANSLWMLSRSSWFPLLISSYFWPFCSASQSPDGWWYFASDWFAPRYSVRALPFTLSNYRRSYEAYLSQCRVDIPSWGTKHPLGMFWHHKVSTLIDAMV SRRVYHTMEKAGQAAWKQVVSEATLSRISSLDVVAHFQHLAAIEETCKYLASRLPMLHNLRMSGSNVTIKYNSTSGRVFAIFPTPDSRPKLHDFQQWLIVVHSSIFSSVAASCTLFVVLWLRVPILRTVFGFHWLGATFLSSSWGSDYKDDDDK, SEQ ID NO: 31: Wherein, amino acids 1-256 are GP2 protein, amino acids 257-258 are linking peptides, amino acids 259-266 are Flag tags, GP3-Flag (MVNSCALLHIFLCCSFLHFICCAMAPSANSTYCFWFPLVRGNFSFELMVNYTVCPPCLTRQAAAQILEPGGSLWCRIGHDRCEESDHDELEFAVPPGLSVEGRLTSVYAWLAFLSFSYTAQFHPEIFGIGNVSEVYVDIQNQLICAVHDGLNATLPSHDNITAVYQSYYQHQVDGGNWFHLEWLRPFFSSWLVLNVSWFLRRSPVSRVSVRVFQTLRPTPPAQQVPLSSKTSAVLGIATRPLRRFAKSLNVARRGSDYKDDDDK, SEQ ID NO: 32: Wherein, amino acids 1-254 are GP3 protein, amino acids 255-256 are linker peptides, and amino acids 257-264 are Flag tags), GP4-Flag (MAASFLFLLVGFERFMVSQAFACKPCFSSSLSDIKTNTTSAASSVVLQNIGCLRHSNPSPAAFRKIPQCRAAIGTPVYITVTANVTDENYLHSSDLLMLSSCLFYASEMSEKGFKVIFGNVSGIVAVCVNFTSYVQHVREFTQRSLVVDHVRLLHFMTPETMRWATVLACLFAILLAIGSDYKDDDDK, SEQ ID NO: 33: Wherein, amino acids 1-178 are GP4 protein, and amino acids 179-180 are linker peptides,The gene encoding the Flag tag (amino acids 181-188) (GP2, GP3, and GP4 proteins are all from the NADC30 strain) was inserted between NheI and NotI in the pCDH-CMV-MCS-EF1-GFP+Puro vector (Ubibio, catalog number VT8070) to obtain the GP2-Flag, GP3-Flag, and GP4-Flag plasmids.

[0127] 24 hours after transfecting 293T cells with plasmids GP2-Flag, GP3-Flag, and GP4-Flag, cells were collected for flow cytometry analysis. First, cells were trypsinized to disperse them into a single-cell suspension. Digestion was terminated with PBS buffer containing 5% FBS, and the supernatant was removed by centrifugation, collecting the cell pellet. Then, 1 mL of fixative was added to fix the cells, and the suspension was incubated at room temperature in the dark for 30 minutes before centrifugation to remove the fixative. Next, the cells were permeabilized twice with 1 mL of 1X permeabilization buffer, with the supernatant removed by centrifugation after each treatment. The cells were then blocked with PBS buffer containing 5% FBS for 30 minutes. After blocking, the blocking solution was removed by centrifugation, and the cells were washed once with 1X permeabilization buffer. The cells were resuspended in 250 µL of 1X permeabilization buffer, and primary antibodies diluted 1:1000 (the genetically engineered antibody (GP4-6 mAb) obtained in step 8 and the Flag antibody (Flag mAb)) were added. The cells were incubated at 4°C in the dark for 60 minutes. After incubation with the primary antibody, cells were washed twice with 1X permeabilization buffer, and then an appropriate amount of secondary antibody (PE Goatanti-mouse IgG, Biolegend, catalog number 405307) was added. Cells were incubated at 4°C in the dark for 30 minutes. After secondary antibody incubation, cells were washed twice with 1X permeabilization buffer. Finally, cells were resuspended in PBS buffer containing 1% FBS for flow cytometry analysis. Results are as follows: Figure 3 As shown: GP4-6 antibody specifically binds to GP4 protein.

[0128] 10. Antibody Affinity Verification The genetically engineered antibody (GP4-6) obtained in step 8 was immobilized on the surface of a CM5 chip using an amine coupling method, with a target immobilization amount of 50-200 RU. Next, using HBS-EP+ as the run buffer, a series of concentration gradients of GP234 antigen were prepared from low to high (1.56 to 50 nM). On the SPR instrument, samples were injected sequentially in ascending order of concentration, with each cycle including brief antigen binding, buffer dissociation, and chip regeneration using glycine (pH 1.5). Finally, after subtracting blank and reference signals using the instrument software, the binding and dissociation curves were globally fitted using a "1:1 binding model" to directly obtain the affinity constant KD value. The results are as follows: Figure 4As shown, its KD is 2.37E-09 M, indicating good affinity.

[0129] The test sample (GP234 complex obtained in step 3) was diluted to 1 μg / mL, and after adding 6× Loading buffer, it was boiled in boiling water for 10 minutes to fully denature it. The gel electrophoresis was performed in constant voltage mode: 100 V for 20 minutes for the stacking gel and 180 V for 40 minutes for the separating gel. Transfer was performed in constant current mode at 200 mA for 2 hours. Blocking buffer (TBS buffer containing 5% skim milk powder) was used for 30 minutes. The primary antibody (the genetically engineered antibody (GP4-6) obtained in step 8, and the control antibody Mouse anti StrepII tag mAb (brand: Abclonal; catalog number: AE066)) was diluted with TBST buffer containing 2.5% skim milk powder, incubated at room temperature for 2 hours, and then washed three times with 0.1% TBST for 10 minutes each time. The secondary antibody (Anti-Mouse IgG (H+L), HRP Conjugate (brand: Promega; catalog number: W4021) was diluted with TBST buffer containing 2.5% skim milk powder. After diluting with buffer and incubating at room temperature for 1 hour, the membrane was washed three times with 0.1% TBST for 10 minutes each time. ECL developing solution was then added for color development. Results are as follows... Figure 5 As shown: GP4-6 antibody can bind to GP4 protein.

[0130] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody against porcine reproductive and respiratory syndrome virus (PRRSV) or an antigen-binding fragment thereof, wherein the antibody against PRSV or an antigen-binding fragment thereof comprises: a1) HCDR1, HCDR2 and HCDR3 included in the heavy chain variable region (VH) of the amino acid sequence shown in SEQ ID NO: 9; and / or LCDR1, LCDR2 and LCDR3 included in the light chain variable region (VL) of the amino acid sequence shown in SEQ ID NO:

10. a2) having one or more amino acid substitutions, deletions, or additions to HCDR1, HCDR2, and HCDR3 compared to those shown in a1); and / or having one or more amino acid substitutions, deletions, or additions to LCDR1, LCDR2, and LCDR3 compared to those shown in a1).

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment includes: b1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 13, HCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 15; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 16, LCDR2 having the amino acid sequence AAT, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 17; b2) VH comprising the following three CDRs: having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 shown in b1); and / or VL comprising the following three CDRs: having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 shown in b1); Wherein, the CDR is defined according to the IMGT numbering system; or The anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment includes: c1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 18, HCDR2 having the amino acid sequence shown in SEQ ID NO: 19, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 20; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 17; or c2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 shown in c1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 shown in c1); Wherein, the CDR is defined according to the Kabat numbering system; or The anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment includes: d1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 23, HCDR2 having the amino acid sequence shown in SEQ ID NO: 24, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 20; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 17; or d2) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 shown in d1); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 shown in d1); Wherein, the CDR is defined according to the Chothia numbering system; or The anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment includes: e1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 25, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 28, LCDR2 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 30; or e2) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 shown in e1); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 shown in e1); The CDR is defined according to the Contact numbering system; Preferably, the heavy chain variable region of the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment further includes the framework region of the heavy chain variable region; Preferably, the light chain variable region of the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment further includes the framework region of the light chain variable region.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The frame region of the heavy chain variable region includes the frame region of the heavy chain variable region of immunoglobulins derived from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese, or a mutant thereof; further including the frame region of the heavy chain variable region of immunoglobulins derived from mice, or a mutant thereof. Preferably, the framework region of the light chain variable region includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; further, it includes the framework region of the light chain variable region or a mutant thereof derived from mouse immunoglobulins. Preferably, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment comprises: The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The antibody against porcine reproductive and respiratory syndrome virus or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese. Preferably, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese. Preferably, the heavy chain constant region includes a heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin; Preferably, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins; Preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Preferably, the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it. Preferably, the antibody against porcine reproductive and respiratory syndrome virus or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; Preferably, the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment is a monoclonal antibody, nanobody, Fab fragment, Fab' fragment, Fab'-SH fragment, F(ab')2 fragment, Fv fragment, single-chain Fv (scFv), dsFv or Fd fragment.

5. A chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises the antibody or an antigen-binding fragment thereof as described in any one of claims 1-4.

6. A multispecific antibody or an antigen-binding fragment thereof, comprising two or more antigen-binding domains, wherein one antigen-binding domain comprises the antibody or an antigen-binding fragment of any one of claims 1-4.

7. Biomaterials, including any one of n1)-n9): n1) A nucleic acid molecule that encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the chimeric antigen receptor of claim 5, or the multispecific antibody or antigen-binding fragment thereof as described in claim 6; n2) An expression cassette containing the nucleic acid molecule described in n1); n3) A carrier containing the nucleic acid molecule described in n1); n4) A carrier containing the expression box described in n2); n5) A cell containing the nucleic acid molecules described in n1); n6) Cells containing the expression cassette described in n2); n7) Cells containing the carrier described in n3); n8) Cells containing the carrier described in n4); n9) A cell comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the chimeric antigen receptor of claim 5, or the multispecific antibody or antigen-binding fragment thereof as described in claim 6; None of the cells described in n5)-n9) contain reproductive material.

8. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the chimeric antigen receptor according to claim 5, or the multispecific antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody is obtained by culturing the cells described in claim 7.

9. A conjugate comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-4 or a multispecific antibody or antigen-binding fragment thereof as claimed in claim 6; and a conjugation portion.

10. The coupling according to claim 9, characterized in that, The coupling portion includes a detectable marker or therapeutic agent; Preferably, the detectable markers include enzymes, radionuclides, fluorescent dyes, luminescent substances, colored substances, and / or biotin; Preferably, the therapeutic agent includes other drugs for the prevention and / or treatment of porcine reproductive and respiratory syndrome virus infection or diseases caused by it.

11. A pharmaceutical composition comprising: The antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the chimeric antigen receptor of claim 5, the multispecific antibody or antigen-binding fragment thereof as described in claim 6, the biomaterial of claim 7, or the conjugate of any one of claims 9-10; and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, characterized in that, The pharmaceutical composition also includes other pharmaceutically active agents; Preferably, the other pharmaceutically active agents include drugs for the prevention and / or treatment of porcine reproductive and respiratory syndrome virus infection or diseases caused by it.

13. Diagnostic or therapeutic reagent kits, comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-4, the chimeric antigen receptor according to claim 5, the multispecific antibody or antigen-binding fragment thereof according to claim 6, the biomaterial according to claim 7, the conjugate according to any one of claims 9-10, or the pharmaceutical composition according to any one of claims 11-12.

14. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the chimeric antigen receptor according to claim 5, the multispecific antibody or antigen-binding fragment thereof according to claim 6, the biomaterial according to claim 7, the conjugate according to any one of claims 9-10, or the pharmaceutical composition according to any one of claims 11-12 in any one of d1)-d6): d1) Prepare products for the diagnosis of porcine reproductive and respiratory syndrome virus infection or diseases caused by it; d2) Prepare products for the prevention and / or treatment of porcine reproductive and respiratory syndrome virus infection or diseases caused by it; d3) Prepare products for detecting the presence or level of porcine reproductive and respiratory syndrome virus or its GP4 protein in samples; d4) Detect the presence or level of porcine reproductive and respiratory syndrome virus or its GP4 protein; d5) Prepare products for drug screening, wherein the drugs are used to prevent and / or treat porcine reproductive and respiratory syndrome virus infection or diseases caused by it; d6) Drug screening, wherein the drug is used to prevent and / or treat porcine reproductive and respiratory syndrome virus infection or diseases caused thereby.

Images